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Genetically Regulated Epigenetic Transcriptional Activation of Retrotransposon Insertion Confers Mouse Dactylaplasia Phenotype

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Genetically Regulated Epigenetic Transcriptional Activation of Retrotransposon Insertion Confers Mouse Dactylaplasia Phenotype Genetically regulated epigenetic transcriptional activation of retrotransposon insertion confers mouse dactylaplasia phenotype Hiroki Kano*, Hiroki Kurahashi†, and Tatsushi Toda*‡ *Division of Clinical Genetics, Department of Medical Genetics, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan; and †Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake 470-1192, Japan Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved September 7, 2007 (received for review June 12, 2007) Dactylaplasia, characterized by missing central digital rays, is an were identified at 10q24 in several SHFM3 families (10–13). The inherited mouse limb malformation that depends on two genetic smallest duplicated region contained a disrupted extra copy of loci. The first locus, Dac, is an insertional mutation around the the dactylin gene and the LBX1, BTRC, POLL, and DPCD genes dactylin gene that is inherited as a semidominant trait. The second in their entirety. The dactylin gene encodes an F-box/WD40 locus is an unlinked modifier, mdac/Mdac, that is polymorphic repeat protein; members of this protein family commonly func- among inbred strains. Mdac dominantly suppresses the dactylapla- tion in ubiquitin-dependent proteolytic pathways (14). Although sia phenotype in mice carrying Dac. However, little is known about the dactylin gene is considered to be the best candidate for either locus or the nature of their interaction. Here we show that SHFM3 and the mouse dactylaplasia phenotype, its specific Dac is a LTR retrotransposon insertion caused by the type D mouse function remains undetermined. In mice, the Dac2J insertion in endogenous provirus element (MusD). This insertion exhibits dif- intron 5 of dactylin results in the absence of the normal dactylin ferent epigenetic states and spatiotemporally expresses depend- mRNA transcript; in contrast, the Dac1J insertion in the up- ing on the mdac/Mdac modifier background. In dactylaplasia mu- stream region affects neither the size nor the amount of dactylin -tants (Dac/؉ mdac/mdac), the LTRs of the insertion contained transcript (3). Furthermore, human SHFM3 patients demon unmethylated CpGs and active chromatin. Furthermore, MusD strate only partial dactylin duplications (10, 11). Therefore, the elements expressed ectopically at the apical ectodermal ridge of possibility exists that the dactylin gene itself is simply a bystander limb buds, accompanying the dactylaplasia phenotype. On the and that Dac insertions have long-range regulatory effects on .other hand, in Dac mutants carrying Mdac (Dac/؉ Mdac/mdac), the neighboring genes 5؅ LTR of the insertion was heavily methylated and enriched with The mouse dactylaplasia phenotype depends not only on the inactive chromatin, correlating with inhibition of the dactylaplasia genotype at the mutated Dac locus but also on homozygosity for phenotype. Ectopic expression was not observed in the presence of a recessive allele in another unlinked locus, mdac, which has Mdac, which we refined to a 9.4-Mb region on mouse chromosome been mapped to chromosome 13, between D13Mit10 and 13. We report a pathogenic mutation caused by MusD. Our findings D13Mit99 (2). This locus is polymorphic among inbred strains, indicate that ectopic expression from the MusD insertion correlates and two alleles have been identified. Inbred strains such as with the dactylaplasia phenotype and that Mdac acts as a defen- BALB/cJ, A/J, and 129/J carry mdac, which permits Dac expres- sive factor to protect the host genome from pathogenic MusD sion; on the other hand, inbred strains such as CBA/J, C3H/J, and insertions. C57BL/6J carry the Mdac allele, which dominantly inactivates Dac (2). The Dac1J and Dac2J alleles are equally sensitive to Mdac dactylin ͉ ectrodactyly ͉ LTR ͉ split hand/split foot malformation ͉ (3). Therefore, the dactylaplasia phenotype is observed in only methylation mice homozygous for mdac (Dac/ϩ mdac/mdac or Dac/Dac mdac/mdac) and is never observed in mice carrying Mdac, actylaplasia is an inherited mouse limb malformation that is regardless of their Dac status (Dac/ϩ Mdac/mdac or Dac/Dac Dcharacterized by missing central digital rays. The Dac mutation Mdac/mdac). Mutational insertions in the Dac locus have been is inherited as a semidominant trait, evidenced by missing central identified and partially cloned; however, little is known about the digits in the fore- and hindlimbs of heterozygous mice and mono- insertional mutation or its modifier. dactyly in homozygous mice (1, 2). The Dac locus has been mapped The present study aimed to characterize Dac, Mdac, and to the distal end of chromosome 19. Two independent Dac mutant interactions between the two loci to elucidate the pathological alleles, Dac1J and Dac2J, arose spontaneously in breeding colonies. mechanism of dactylaplasia. Our study found that both Dac Both are insertions residing within the same locus: Dac1J is located in the region upstream of the dactylin gene, and Dac2J is located in intron 5 of dactylin (3). Southern blot analysis indicated that both Author contributions: H. Kano, H. Kurahashi, and T.T. designed research; H. Kano per- formed research; H. Kano analyzed data; and H. Kano and T.T. wrote the paper. insertions are larger than 4.5 kb; however, ‘‘jumping PCR’’ identi- fied only the LTR portion of the insertion (3). Partial PCR products The authors declare no conflict of interest. terminating in the 5Ј and 3Ј LTRs cross-prime each other, resulting This article is a PNAS Direct Submission. in amplified products that lack any of the internal sequence between Abbreviations: AER, apical ectodermal ridge; E, embryonic day; ETn, early transposon; MusD, the type D mouse endogenous provirus element; SHFM, split hand/split foot LTRs. Therefore, these insertions were thought to be caused by an malformation. early transposon (ETn) element, which is a common mutagen in Data deposition: The sequences reported in this paper have been deposited in the DNA mice (4, 5). Data Bank of Japan (accession nos. AB305072 and AB305073). Split hand/split foot malformation (SHFM) in humans is a See Commentary on page 18879. congenital limb malformation that has an ectrodactyly pheno- ‡To whom correspondence should be addressed at: Division of Clinical Genetics, Depart- type analogous to that of the dactylaplasia mouse. SHFM is ment of Medical Genetics, Osaka University Graduate School of Medicine, 2-2-B9, Yamad- genetically heterogeneous; to date, five different loci, SHFM1 to aoka, Suita, Osaka 565-0871, Japan. E-mail: [email protected]. SHFM5, have been mapped. Dactylaplasia is a mouse model of This article contains supporting information online at www.pnas.org/cgi/content/full/ SHFM3 because the Dac locus is syntenic to the SHFM3 locus 0705483104/DC1. (10q24) (2, 6–9). Recently, 0.5-Mb tandem genomic duplications © 2007 by The National Academy of Sciences of the USA 19034–19039 ͉ PNAS ͉ November 27, 2007 ͉ vol. 104 ͉ no. 48 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0705483104 Downloaded by guest on September 30, 2021 A Fgf8 dactylin exon 1 2 3 4 5 6 7 8 9 SEE COMMENTARY Tel Cen Dac1J insertion 5‘ LTR Dac2J insertion 3‘ LTR GTTATG GTTATG GAGAAA GAGAAA 3‘ LTR 5‘ LTR exon 1 2 3 4 5 6 7 8 9 Tel Cen B Dac1J locus Dac2J locus C orp 5‘ LTR gag pol 3‘ LTR 2J 1J Dac Dac / / 2J 1J O O 2 2 M2 H M1 +/+ +/+ H Dac Dac (bp) (bp) 23,130 ,9 416 (AF246633) 6,557 4,361 MusD 0,1 00 850 650 500 Dac1J insertion Fig. 1. Characterization of Dac insertions. (A) Insertions of LTR retrotransposons around the dactylin gene. The Dac1J insertion was integrated 10 kb upstream of the dactylin gene in antisense orientation. The Dac2J insertion was integrated into intron 5 of the dactylin gene in sense orientation. (B) PCR amplification of the insertions. The Dac1J and Dac2J insertions were 7,486 and 7,473 bp, respectively, and were 99.6% identical in sequence. M1, Lambda DNA-HindIII digest (New England Biolabs, Beverly, MA); M2, 1Kb Plus DNA Ladder (Invitrogen). (C) Dot plot DNA comparison of the Dac1J insertion and the MusD element (AF246633). The stringency of comparison was 19 of 23. Each Dac insertion shared 100% identity within the 5Ј and 3Ј LTRs and contained intact ORFs for the gag, pro, and pol genes. insertions are caused by a type D mouse endogenous provirus their identical genotypes, suggesting an epigenetic effect on (MusD) element. We observed a correlation between the dac- phenotype severity. To investigate the epigenetic status of these tylaplasia phenotype and the epigenetic status of the MusD insertions, bisulfite sequencing and ChIP studies were per- insertion, which is modulated by its modifier. Furthermore, we formed for the Dac1J insertion by using freshly prepared em- show ectopic expression of MusD and its related elements in Dac bryonic tissues. An unrelated MusD element (AL773522) in the mutant limb buds. These observations demonstrate the impact of mouse genome was used as a control for each experiment. This retrotransposon insertions in the genome, as well as a host element shows the greatest sequence similarity to Dac insertions defensive mechanism against retrotransposons. in the mouse genome; its 5Ј and 3Ј LTRs are 100% identical, containing 18 CpGs, but it has an ORF-disrupting mutation in Results the pol gene due to a 1-bp deletion. It is known that most LTR Characterization of Dac Insertions. Two independent, spontane- 1J 2J retrotransposons in somatic cells are maintained in a heavily ously arising Dac mutant alleles, Dac and Dac , are caused by methylated and silent state (16). As expected, bisulfite sequenc- insertions around the dactylin gene. Each was previously par- ing showed that both the 5Ј and 3Ј LTRs of the AL773522 tially cloned (3). To further characterize these insertions, we element were heavily methylated in Dac mutant mice (Dac1J/ϩ GENETICS isolated each by PCR and sequenced them directly (Fig.
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